METHOD FOR PRODUCING AN L-AMINO ACID USING A BACTERIUM OF THE FAMILY ENTEROBACTERIACEAE HAVING ATTENUATED EXPRESSION OF THE yjjK GENE

ABSTRACT

The present invention provides a method for producing L-amino acids by fermentation using a bacterium of the family Enterobacteriaceae, particularly a bacterium belonging to the genus  Escherichia , which has been modified to attenuate expression of the yjjK gene.

This application claims priority under 35 U.S.C. §119 to Russian PatentApplication No. 2013118637, filed Apr. 23, 2013, the entirety of whichis incorporated by reference herein. Also, the Sequence Listing filedelectronically herewith is hereby incorporated by reference (File name:2014-04-23T_US-495 Seq List; File size: 21 KB; Date recorded: Apr. 23,2014).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the microbiological industry, andspecifically to a method for producing L-amino acids by fermentation ofa bacterium of the family Enterobacteriaceae which has been modified toattenuate expression of the yjjK gene.

2. Description of the Related Art

Conventionally, L-amino acids are industrially produced by fermentationmethods utilizing strains of microorganisms obtained from naturalsources, or mutants thereof. Typically, the microorganisms are modifiedto enhance production yields of L-amino acids.

Many techniques to enhance L-amino acid production yields have beenreported, including transformation of microorganisms with recombinantDNA (see, for example, U.S. Pat. No. 4,278,765 A) and alteration ofregulatory regions such as promoter, leader sequence, and/or attenuator,or others known to the person skilled in the art (see, for example,US20060216796 A1 and WO9615246 A1). Other techniques for enhancingproduction yields include increasing the activities of enzymes involvedin amino acid biosynthesis and/or desensitizing the target enzymes tothe feedback inhibition by the resulting L-amino acid (see, for example,WO9516042 A1, EP0685555 A1 or U.S. Pat. Nos. 4,346,170 A, 5,661,012 A,and 6,040,160 A).

Another method for enhancing L-amino acids production yields is toattenuate expression of a gene or several genes which are involved indegradation of the target L-amino acid, genes which divert theprecursors of the target L-amino acid from the L-amino acid biosyntheticpathway, genes involved in the redistribution of the carbon, nitrogen,and phosphate fluxes, and genes encoding toxins, etc.

The yjjK gene product has been identified using two-dimensional gelelectrophoresis (Link A. J. et al., Comparing the predicted and observedproperties of proteins encoded in the genome of Escherichia coli K-12,Electrophoresis, 1997, 18(8):1259-1313). The YjjK protein wascharacterized later as a putative ABC transporter (ATP bindingcassette), a member of one of the largest protein families known. ABCtransporters have the common distinctive architecture, which consists oftwo transmembrane domains (TMDs) embedded in the membrane bilayer andtwo nucleotide-binding domains (NBDs) located in the cytoplasm (Rees D.C. et al., ABC transporters: the power to change, Nat. Rev. Mol. CellBiol., 2009, 10(3): 218-227). The prokaryotic ABC transporters canrecruit a binding protein to translocate substrates. ATP binding to thenucleotide-binding domains and hydrolysis drive the conformationalchanges that result in translocation of a substrate. Functioning asimporters or exporters, ABC transporters can transport a wide variety ofsubstrates which include ions, toxins, antibiotics, lipids,polysaccharides, nutrients such as amino acids, peptides, sugars, and soforth. Little is known about function of the YjjK protein (Linton K. J.and Higgins C. F., The Escherichia coli ATP-binding cassette (ABC)proteins, Mol. Microbiol., 1998, 28(1):5-13). It is only predicted bythe sequence similarity approach that this protein is the ATP-bindingcomponent of a member of the ABC superfamily, subfamily 3 oftransporters having the NBD-NBD domain organization.

Until now, no data has been reported demonstrating the effect fromattenuating the yjjK gene on L-amino acid production by the modifiedbacterial strains of the family Enterobacteriaceae.

SUMMARY OF THE INVENTION

An aspect of the present invention is to provide a bacterium belongingto the family Enterobacteriaceae, which can belong to the genusEscherichia and, more specifically, to the species Escherichia coli (E.coli), which has been modified to attenuate expression of the yjjK gene.

Another aspect of the present invention is to provide a method forproducing L-amino acids such as L-alanine, L-arginine, L-asparagine,L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine,glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine,L-tryptophan, L-tyrosine, and L-valine using a bacterium of the familyEnterobacteriaceae as described hereinafter.

These aims were achieved by the unexpected finding that attenuation ofexpression of the yjjK gene, in particular inactivation of the yjjKgene, on the chromosome of a bacterium belonging to the familyEnterobacteriaceae, which can belong to the genus Escherichia and, morespecifically, to the species Escherichia coli, confers on themicroorganism higher productivity of L-amino acids, in particular, butare not limited to L-valine and L-histidine.

An aspect of the present invention is to provide a method for producingan L-amino acid comprising:

(i) cultivating the bacterium of the family Enterobacteriaceae in aculture medium to produce said L-amino acid in the bacterium and/or theculture medium; and

(ii) collecting said L-amino acid from the bacterium and/or the culturemedium, wherein the bacterium has been modified to attenuate expressionof the yjjK gene.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the bacterium belongs to the genus Escherichia.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the bacterium belongs to the speciesEscherichia coli.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the bacterium belongs to the genus Pantoea.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the bacterium belongs to the species Pantoeaananatis.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the expression is attenuated due toinactivation of the yjjK gene.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the gene is deleted.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the yjjK gene is encoded by the nucleotidesequence of SEQ ID NO: 1 or a variant nucleotide sequence of SEQ ID NO:1.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the L-amino acid is selected from the groupconsisting of an aromatic L-amino acid and a non-aromatic L-amino acid.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the aromatic L-amino acid is selected from thegroup consisting of L-phenylalanine, L-tryptophan, and L-tyrosine.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the non-aromatic L-amino acid is selected fromthe group consisting of L-alanine, L-arginine, L-asparagine, L-asparticacid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine,L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-ornithine, L-proline, L-serine, L-threonine, and L-valine.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is described in detail below.

1. Bacterium

The phrase “an L-amino acid-producing bacterium” can mean a bacterium ofthe family Enterobacteriaceae which has an ability to produce, excreteor secrete, and/or cause accumulation of an L-amino acid in a culturemedium or the bacterial cells when the bacterium is cultured in themedium.

The phrase “an L-amino acid-producing bacterium” can also mean abacterium which is able to produce, excrete or secrete, and/or causeaccumulation of an L-amino acid in a culture medium in an amount largerthan a wild-type or parental strain, such as E. coli K-12, and can meanthat the microorganism is able to cause accumulation in a medium of anamount not less than 0.5 g/L or not less than 1.0 g/L of the targetL-amino acid. The bacterium can produce an amino acid either alone or asa mixture of two or more kinds of amino acids.

The phrase “L-amino acid-producing ability” can mean the ability of thebacterium to produce, excrete or secrete, and/or cause accumulation ofthe L-amino acid in a medium or the bacterial cells to such a level thatthe L-amino acid can be collected from the medium or the bacterialcells, when the bacterium is cultured in the medium.

The phrase “L-amino acid” can mean L-alanine, L-arginine, L-asparagine,L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine,glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine,L-tryptophan, L-tyrosine, and L-valine.

The phrase “aromatic L-amino acid” includes, for example,L-phenylalanine, L-tryptophan, and L-tyrosine.

The phrase “non-aromatic L-amino acid” includes, for example, L-alanine,L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine,L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine,L-leucine, L-lysine, L-methionine, L-ornithine, L-proline, L-serine,L-threonine, and L-valine.

An L-amino acid can belong to one or more L-amino acid families. As anexample, the L-amino acid can belong to the glutamate family includingL-arginine, L-glutamic acid, L-glutamine, and L-proline; the serinefamily including L-cysteine, glycine, and L-serine; the aspartate familyincluding L-asparagine, L-aspartic acid, L-isoleucine, L-lysine,L-methionine, and L-threonine; the pyruvate family including L-alanine,L-isoleucine, L-valine, and L-leucine; and the aromatic family includingL-phenylalanine, L-tryptophan, and L-tyrosine.

L-Arginine, L-cysteine, L-glutamic acid, L-histidine, L-leucine,L-lysine, L-phenylalanine, L-proline, L-threonine, L-tryptophan, andL-valine are particular examples. L-Histidine and L-valine arepreferable examples.

The bacteria belonging to the family Enterobacteriaceae can be from thegenera Enterobacter, Erwinia, Escherichia, Klebsiella, Morganella,Pantoea, Photorhabdus, Providencia, Salmonella, Yersinia, and so forth,and can have the ability to produce an L-amino acid. Specifically, thoseclassified into the family Enterobacteriaceae according to the taxonomyused in the NCBI (National Center for Biotechnology Information)database (www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=543) canbe used. Examples of strains from the family Enterobacteriaceae whichcan be modified include a bacterium of the genus Escherichia,Enterobacter or Pantoea.

Strains of Escherichia bacterium which can be modified to obtainEscherichia bacteria in accordance with the presently disclosed subjectmatter are not particularly limited, and specifically, those describedin the work of Neidhardt et al. can be used (Bachmann, B. J.,Derivations and genotypes of some mutant derivatives of E. coli K-12, p.2460-2488. In F. C. Neidhardt et al. (ed.), E. coli and Salmonella:cellular and molecular biology, 2^(nd) ed. ASM Press, Washington, D.C.,1996). The species E. coli is a particular example. Specific examples ofE. coli include E. coli W3110 (ATCC 27325), E. coli MG1655 (ATCC 47076),and so forth, which are derived from the prototype wild-type strain, E.coli K-12 strain. These strains are available from, for example, theAmerican Type Culture Collection (P.O. Box 1549, Manassas, Va. 20108,United States of America). That is, registration numbers are given toeach of the strains, and the strains can be ordered by using theseregistration numbers (refer to www.atcc.org). The registration numbersof the strains are listed in the catalogue of the American Type CultureCollection.

Examples of the Enterobacter bacteria include Enterobacter agglomerans,Enterobacter aerogenes, and so forth. Examples of the Pantoea bacteriainclude Pantoea ananatis, and so forth. Some strains of Enterobacteragglomerans were recently reclassified into Pantoea agglomerans, Pantoeaananatis or Pantoea stewartii on the basis of nucleotide sequenceanalysis of 16S rRNA, etc. A bacterium belonging to any of the genusEnterobacter or Pantoea may be used so long as it is a bacteriumclassified into the family Enterobacteriaceae. When a Pantoea ananatisstrain is bred by genetic engineering techniques, Pantoea ananatisAJ13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ13601strain (FERM BP-7207) and derivatives thereof can be used. These strainswere identified as Enterobacter agglomerans when they were isolated, anddeposited as Enterobacter agglomerans. However, they were recentlyre-classified as Pantoea ananatis on the basis of nucleotide sequencingof 16S rRNA and so forth as described above.

L-Amino Acid-Producing Bacteria

The bacterium of the present invention belonging to the familyEnterobacteriaceae and modified to attenuate expression of the yjjKgene, which is able to produce either an aromatic or a non-aromaticL-amino acid, can be used.

The bacterium may inherently have the L-amino acid-producing ability ormay be modified to have an L-amino acid-producing ability by using amutation method or DNA recombination techniques. The bacterium can beobtained by attenuating expression of the yjjK gene in a bacterium whichinherently has the ability to produce L-amino acids. Alternatively, thebacterium can be obtained by imparting the ability to produce L-aminoacids to a bacterium already having the attenuated expression of theyjjK gene.

L-Arginine-Producing Bacteria

Examples of parental strains which can be used to deriveL-arginine-producing bacteria include, but are not limited to strainsbelonging to the genus Escherichia such as E. coli strain 237 (VKPMB-7925) (U.S. Patent Application 2002/058315 A1) and its derivativestrains harboring mutant N-acetylglutamate synthase (RU2215783), E. colistrain 382 (VKPM B-7926) (EP1170358 A1), an arginine-producing straininto which argA gene encoding N-acetylglutamate synthetase is introducedtherein (EP1170361 A1), and the like.

Examples of parental strains which can be used to deriveL-arginine-producing bacteria also include strains in which expressionof one or more genes encoding an L-arginine biosynthetic enzyme areenhanced. Examples of such genes include genes encodingN-acetyl-γ-glutamylphosphate reductase (argC), ornithineacetyltransferase (argJ), N-acetylglutamate kinase (argB),N-acetylornithine aminotransferase (argD), ornithinecarbamoyltransferase (argF), argininosuccinate synthase (argG),argininosuccinate lyase (argH), and carbamoyl phosphate synthetase(carAB).

L-Citrulline-Producing Bacteria

Examples of parental strains which can be used to deriveL-citrulline-producing bacteria include, but are not limited to strainsbelonging to the genus Escherichia such as E. coli mutantN-acetylglutamate synthase strains 237/pMADS 11, 237/pMADS 12, and237/pMADS 13 (RU2215783 C2, EP1170361 B1, U.S. Pat. No. 6,790,647 B2),E. coli strains 333 (VKPM B-8084) and 374 (VKPM B-8086), both harboringmutant feedback-resistant carbamoyl phosphate synthetase (Russian PatentRU2264459 C2), strains E. coli, in which α-ketoglutarate synthaseactivity is increased, and ferredoxin NADP⁺ reductase, pyruvate synthaseor α-ketoglutarate dehydrogenase activities are additionally modified(EP2133417 A1), and strain P. ananantis NA1sucAsdhA, in which succinatedehydrogenase and α-ketoglutarate dehydrogenase activities are decreased(US Patent Application No 2009286290), and the like.

As L-citrulline is an intermediate of L-arginine biosynthetic pathway,examples of parent strains, which can be used to deriveL-citrulline-producing bacteria, include strains, in which expression ofone or more genes encoding an L-arginine biosynthetic enzyme isenhanced. Examples of such genes include, but are not limited to, genesencoding N-acetylglutamate synthase (argA), N-acetylglutamate kinase(argB), N-acetylglutamyl phosphate reductase (argC), acetylornithinetransaminase (argD), acetylornithine deacetylase (argE), ornithinecarbamoyltransferase (argF/I), and carbamoyl phosphate synthetase(carAB), or combinations thereof.

L-citrulline-producing bacterium can be also easily obtained from anyL-arginine-producing bacterium, for example E. coli 382 stain (VKPMB-7926), by inactivation of argininosuccinate synthase encoded by argGgene.

L-Cysteine-Producing Bacteria

Examples of parental strains which can be used to deriveL-cysteine-producing bacteria include, but are not limited to strainsbelonging to the genus Escherichia such as E. coli JM15 which istransformed with different cysE alleles encoding feedback-resistantserine acetyltransferases (U.S. Pat. No. 6,218,168, Russian Patent No.2279477), E. coli W3110 having overexpressed genes which encode proteinssuitable for secreting substances toxic for cells (U.S. Pat. No.5,972,663), E. coli strains having lowered cysteine desulfhydraseactivity (JP11155571 A2), E. coli W3110 with increased activity of apositive transcriptional regulator for cysteine regulon encoded by thecysB gene (WO0127307 A1), E. coli JM15(ydeD) (U.S. Pat. No. 6,218,168),and the like.

L-Glutamic Acid-Producing Bacteria

Examples of parental strains which can be used to derive L-glutamicacid-producing bacteria include, but are not limited to strainsbelonging to the genus Escherichia such as E. coli VL334thrC⁺ (EP1172433). The E. coli VL334 (VKPM B-1641) is an L-isoleucine andL-threonine auxotrophic strain having mutations in thrC and ilvA genes(U.S. Pat. No. 4,278,765). A wild-type allele of the thrC gene wastransferred by the method of general transduction using a bacteriophageP1 grown on the wild-type E. coli strain K12 (VKPM B-7) cells. As aresult, an L-isoleucine auxotrophic strain VL334thrC⁺ (VKPM B-8961),which is able to produce L-glutamic acid, was obtained.

Examples of parental strains which can be used to derive the L-glutamicacid-producing bacteria include, but are not limited to strains in whichexpression of one or more genes encoding an L-glutamic acid biosyntheticenzyme are enhanced. Examples of such genes include genes encodingglutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamatesynthetase (gltAB), isocitrate dehydrogenase (icdA), aconitate hydratase(acnA, acnB), citrate synthase (gltA), phosphoenolpyruvate carboxylase(ppc), pyruvate carboxylase (pyc), pyruvate dehydrogenase (aceEF, lpdA),pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA),enolase (eno), phosphoglyceromutase (pgmA, pgmI), phosphoglyceratekinase (pgk), glyceraldehyde-3-phosphate dehydrogenase (gapA), triosephosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp),phosphofructokinase (pfkA, pfkB), and glucose phosphate isomerase (pgi).

Examples of strains modified so that expression of the citratesynthetase gene, the phosphoenolpyruvate carboxylase gene, and/or theglutamate dehydrogenase gene is/are enhanced include those disclosed inEP1078989 A2, EP955368 A2, and EP952221 A2.

Examples of parental strains which can be used to derive the L-glutamicacid-producing bacteria also include strains having decreased oreliminated activity of an enzyme that catalyzes synthesis of a compoundother than L-glutamic acid by branching off from an L-glutamic acidbiosynthesis pathway. Examples of such enzymes include isocitrate lyase(aceA), α-ketoglutarate dehydrogenase (sucA), phosphotransacetylase(pta), acetate kinase (ack), acetohydroxy acid synthase (ilvG),acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactatedehydrogenase (ldh), and glutamate decarboxylase (gadAB). Bacteriabelonging to the genus Escherichia deficient in the α-ketoglutaratedehydrogenase activity or having a reduced α-ketoglutarate dehydrogenaseactivity and methods for obtaining them are described in U.S. Pat. Nos.5,378,616 and 5,573,945. Specifically, these strains include thefollowing:

E. coli W3110sucA::Km^(R)

E. coli AJ12624 (FERM BP-3853)

E. coli AJ12628 (FERM BP-3854)

E. coli AJ12949 (FERM BP-4881)

E. coli W3110sucA::Km^(R) is a strain obtained by disrupting theα-ketoglutarate dehydrogenase gene (hereinafter referred to as “sucAgene”) of E. coli W3110. This strain is completely deficient inα-ketoglutarate dehydrogenase.

Other examples of L-glutamic acid-producing bacterium include thosewhich belong to the genus Escherichia and have resistance to an asparticacid antimetabolite. These strains can also be deficient in theα-ketoglutarate dehydrogenase activity and include, for example, E. coliAJ13199 (FERM BP-5807) (U.S. Pat. No. 5,908,768), FFRM P-12379, whichadditionally has a low L-glutamic acid decomposing ability (U.S. Pat.No. 5,393,671), AJ13138 (FERM BP-5565) (U.S. Pat. No. 6,110,714), andthe like.

Examples of L-glutamic acid-producing bacteria include mutant strainsbelonging to the genus Pantoea which are deficient in theα-ketoglutarate dehydrogenase activity or have a decreasedα-ketoglutarate dehydrogenase activity, and can be obtained as describedabove. Such strains include Pantoea ananatis AJ13356. (U.S. Pat. No.6,331,419). Pantoea ananatis AJ13356 was deposited at the NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology, Ministry of International Trade and Industry(currently, Incorporated Administrative Agency, National Institute ofTechnology and Evaluation, International Patent Organism (NITE IPOD),#120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken 292-0818, JAPAN) onFeb. 19, 1998 under an accession number of FERM P-16645. It was thenconverted to an international deposit under the provisions of BudapestTreaty on Jan. 11, 1999 and received an accession number of FERMBP-6615. Pantoea ananatis AJ13356 is deficient in α-ketoglutaratedehydrogenase activity as a result of disruption of the αKGDH-E1 subunitgene (sucA). The above strain was identified as Enterobacter agglomeranswhen it was isolated and deposited as the Enterobacter agglomeransAJ13356. However, it was recently re-classified as Pantoea ananatis onthe basis of nucleotide sequencing of 16S rRNA and so forth. AlthoughAJ13356 was deposited at the aforementioned depository as Enterobacteragglomerans, for the purposes of this specification, they are describedas Pantoea ananatis.

L-Histidine-Producing Bacteria

Examples of parental strains which can be used to deriveL-histidine-producing bacteria include, but are not limited to strainsbelonging to the genus Escherichia such as E. coli strain 24 (VKPMB-5945, RU2003677 C1), E. coli strain 80 (VKPM B-7270, RU2119536 C1), E.coli NRRL B-12116-B12121 (U.S. Pat. No. 4,388,405), E. coli H-9342 (FERMBP-6675) and H-9343 (FERM BP-6676) (U.S. Pat. No. 6,344,347), E. coliH-9341 (FERM BP-6674) (EP1085087), E. coli AI80/pFM201 (U.S. Pat. No.6,258,554), and the like.

Examples of parental strains which can be used to deriveL-histidine-producing bacteria also include strains in which expressionof one or more genes encoding an L-histidine biosynthetic enzyme areenhanced. Examples of such genes include genes encoding ATPphosphoribosyltransferase (hisG), phosphoribosyl-AMP cyclohydrolase(hisI), phosphoribosyl-AMP cyclohydrolase/phosphoribosyl-ATPpyrophosphatase (hisIE), phosphoribosylformimino-5-aminoimidazolecarboxamide ribotide isomerase (hisA), amidotransferase (hisH),histidinol phosphate aminotransferase (hisC), histidinol phosphatase(hisB), histidinol dehydrogenase (hisD), and so forth.

It is known that the L-histidine biosynthetic enzymes encoded by hisGand hisBHAFI are inhibited by L-histidine, and therefore anL-histidine-producing ability can also be efficiently enhanced byintroducing a mutation conferring resistance to the feedback inhibitioninto ATP phosphoribosyltransferase (Russian Patent Nos. 2003677 and2119536).

Specific examples of strains having an L-histidine-producing abilityinclude E. coli FERM-P 5038 and 5048 which have been transformed with avector carrying a DNA encoding an L-histidine-biosynthetic enzyme (JP56-005099 A), E. coli strains transformed with rht, a gene for an aminoacid-export (EP1016710 A), E. coli 80 strain imparted withsulfaguanidine, DL-1,2,4-triazole-3-alanine, and streptomycin-resistance(VKPM B-7270, RU2119536), and E. coli MG1655+hisGr hisL′_Δ ΔpurR(RU2119536 C1; Doroshenko V. G. et al., The directed modification ofEscherichia coli MG1655 to obtain histidine-producing mutants, Prikl.Biochim. Mikrobiol. (Russian), 2013, 49(2):149-154), and so forth.

L-Isoleucine-Producing Bacteria

Examples of parental strains which can be used to deriveL-isoleucine-producing bacteria include, but are not limited to mutantshaving resistance to 6-dimethylaminopurine (JP 5-304969 A), mutantshaving resistance to an isoleucine analogue such as thiaisoleucine andisoleucine hydroxamate, and mutants additionally having resistance toDL-ethionine and/or arginine hydroxamate (JP 5-130882 A). In addition,recombinant strains transformed with genes encoding proteins involved inL-isoleucine biosynthesis, such as threonine deaminase andacetohydroxate synthase, can also be used as parental strains (JP 2-458A, EP0356739 A1, and U.S. Pat. No. 5,998,178).

L-Leucine-Producing Bacteria

Examples of parental strains which can be used to deriveL-leucine-producing bacteria include, but are not limited to strainsbelonging to the genus Escherichia such as E. coli strains resistant toleucine (for example, the strain 57 (VKPM B-7386, U.S. Pat. No.6,124,121)) or leucine analogs including β-2-thienylalanine,3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine (JP 62-34397 Band JP 8-70879 A); E. coli strains obtained by the gene engineeringmethod described in WO96/06926; E. coli H-9068 (JP 8-70879 A), and thelike.

The bacterium can be improved by enhancing the expression of one or moregenes involved in L-leucine biosynthesis. Examples include genes of theleuABCD operon, which can be represented by a mutant leuA gene encodingisopropylmalate synthase freed from feedback inhibition by L-leucine(U.S. Pat. No. 6,403,342). In addition, the bacterium can be improved byenhancing the expression of one or more genes encoding proteins whichexcrete L-amino acid from the bacterial cell. Examples of such genesinclude the b2682 and b2683 genes (ygaZH genes) (EP1239041 A2).

L-Lysine-Producing Bacteria

Examples of L-lysine-producing bacteria belonging to the familyEscherichia include mutants having resistance to an L-lysine analogue.The L-lysine analogue inhibits growth of bacteria belonging to the genusEscherichia, but this inhibition is fully or partially desensitized whenL-lysine is present in the medium. Examples of the L-lysine analogueinclude, but are not limited to oxalysine, lysine hydroxamate,S-(2-aminoethyl)-L-cysteine (AEC), γ-methyllysine, α-chlorocaprolactam,and so forth. Mutants having resistance to these lysine analogues can beobtained by subjecting bacteria belonging to the genus Escherichia to aconventional artificial mutagenesis treatment. Specific examples ofbacterial strains useful for producing L-lysine include E. coli AJ11442(FERM BP-1543, NRRL B-12185; see U.S. Pat. No. 4,346,170) and E. coliVL611. In these microorganisms, feedback inhibition of aspartokinase byL-lysine is desensitized.

Examples of parental strains which can be used to deriveL-lysine-producing bacteria also include strains in which expression ofone or more genes encoding an L-lysine biosynthetic enzyme are enhanced.Examples of such genes include, but are not limited to genes encodingdihydrodipicolinate synthase (dapA), aspartokinase (lysC),dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase(lysA), diaminopimelate dehydrogenase (ddh) (U.S. Pat. No. 6,040,160),phosphoenolpyruvate carboxylase (ppc), aspartate semialdehydedehydrogenease (asd), and aspartase (aspA) (EP1253195 A1). In addition,the parental strains may have an increased level of expression of thegene involved in energy efficiency (cyo) (EP1170376 A1), the geneencoding nicotinamide nucleotide transhydrogenase (pntAB) (U.S. Pat. No.5,830,716), the ybjE gene (WO2005/073390), or combinations thereof.

L-Amino acid-producing bacteria may have reduced or no activity of anenzyme that catalyzes a reaction which causes a branching off from theL-amino acid biosynthesis pathway and results in the production ofanother compound. Also, the bacteria may have reduced or no activity ofan enzyme that negatively acts on L-amino acid synthesis oraccumulation. Examples of such enzymes involved in L-lysine productioninclude homoserine dehydrogenase, lysine decarboxylase (cadA, ldcC),malic enzyme, and so forth, and strains in which activities of theseenzymes are decreased or deleted are disclosed in WO95/23864,WO96/17930, WO2005/010175, and so forth.

Expression of both the cadA and ldcC genes encoding lysine decarboxylasecan be decreased in order to decrease or delete the lysine decarboxylaseactivity. Expression of the both genes can be decreased by, for example,the method described in WO2006/078039.

Examples of L-lysine-producing bacteria can include the E. coliWC196ΔcadAΔldcC/pCABD2 strain (WO2006/078039). The strain wasconstructed by introducing the plasmid pCABD2 containing lysinebiosynthesis genes (U.S. Pat. No. 6,040,160) into the WC196 strainhaving disrupted cadA and ldcC genes which encode lysine decarboxylase.

The WC196 strain was bred from the W3110 strain, which was derived fromE. coli K-12 by replacing the wild-type lysC gene on the chromosome ofthe W3110 strain with a mutant lysC gene encoding a mutant aspartokinaseIII in which threonine at position 352 was replaced with isoleucine,resulting in desensitization of the feedback inhibition by L-lysine(U.S. Pat. No. 5,661,012), and conferring AEC resistance to theresulting strain (U.S. Pat. No. 5,827,698). The WC196 strain wasdesignated E. coli AJ13069, deposited at the National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology (currently Incorporated Administrative Agency, NationalInstitute of Technology and Evaluation, International Patent Organism(NITE IPOD), #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken292-0818, JAPAN) on Dec. 6, 1994, and assigned an accession number ofFERM P-14690. Then, it was converted to an international deposit underthe provisions of the Budapest Treaty on Sep. 29, 1995, and assigned anaccession number of FERM BP-5252 (U.S. Pat. No. 5,827,698).

The WC196ΔcadAΔldcC strain itself is also an exemplaryL-lysine-producing bacterium. The WC196ΔcadAΔldcC was designatedAJ110692 and deposited at the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology (currentlyIncorporated Administrative Agency, National Institute of Technology andEvaluation, International Patent Organism (NITE IPOD), #120, 2-5-8Kazusakamatari, Kisarazu-shi, Chiba-ken 292-0818, JAPAN) on Oct. 7, 2008as an international deposit under an accession number of FERM BP-11027.

L-Methionine-Producing Bacteria

Examples of L-methionine-producing bacteria and parent strains which canbe used to derive L-methionine-producing bacteria include, but are notlimited to Escherichia bacteria strains such as strains AJ11539 (NRRLB-12399), AJ11540 (NRRL B-12400), AJ11541 (NRRL B-12401), AJ 11542 (NRRLB-12402) (GB Patent GB2075055); strains 218 (VKPM B-8125) (Russianpatent RU2209248 C2) and 73 (VKPM B-8126) (Russian patent RU2215782 C2)resistant to norleucine, the L-methionine analog, or the like. Thestrain E. coli 73 has been deposited in the Russian National Collectionof Industrial Microorganisms (VKPM) (Russian Federation, 117545 Moscow,1^(st) Dorozhny proezd, 1) on May 14, 2001 under accession number VKPMB-8126, and was converted to an international deposit under the BudapestTreaty on Feb. 1, 2002. Furthermore, a methionine repressor-deficientstrain and recombinant strains transformed with genes encoding proteinsinvolved in L-methionine biosynthesis such as homoserinetranssuccinylase and cystathionine γ-synthase (JP 2000-139471 A) canalso be used as parent strains.

L-Ornithine-Producing Bacteria

L-ornithine-producing bacterium can be easily obtained from anyL-arginine-producing bacterium, for example E. coli 382 stain (VKPMB-7926), by inactivation of ornithine carbamoyltransferase encoded byboth argF and argI genes. Methods for inactivation of ornithinecarbamoyltransferase are described herein.

L-Phenylalanine-Producing Bacteria

Examples of parental strains which can be used to deriveL-phenylalanine-producing bacteria include, but are not limited tostrains belonging to the genus Escherichia such as E. coli AJ12739(tyrA::Tn10, tyrR) (VKPM B-8197), E. coli HW1089 (ATCC 55371) harboringthe mutant pheA34 gene (U.S. Pat. No. 5,354,672), E. coli MWEC101-b(KR8903681), E. coli NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRLB-12147 (U.S. Pat. No. 4,407,952). Also, E. coli K-12 [W3110(tyrA)/pPHAB (FERM BP-3566), E. coli K-12 [W3110 (tyrA)/pPHAD] (FERMBP-12659), E. coli K-12 [W3110 (tyrA)/pPHATerm] (FERM BP-12662), and E.coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB] named as AJ 12604 (FERMBP-3579) may be used as a parental strain (EP488424 B1). Furthermore,L-phenylalanine-producing bacteria belonging to the genus Escherichiawith an enhanced activity of the protein encoded by the yedA gene or theyddG gene may also be used (U.S. patent applications 2003/0148473 A1 and2003/0157667 A1).

L-Proline-Producing Bacteria

Examples of parental strains which can be used to deriveL-proline-producing bacteria include, but are not limited to strainsbelonging to the genus Escherichia such as E. coli 702ilvA (VKPM B-8012)which is deficient in the ilvA gene and is able to produce L-proline(EP1172433 A1). The bacterium can be improved by enhancing theexpression of one or more genes involved in L-proline biosynthesis.Examples of genes which can be used in L-proline-producing bacteriainclude the proB gene encoding glutamate kinase with desensitizedfeedback inhibition by L-proline (DE3127361 A1). In addition, thebacterium can be improved by enhancing the expression of one or moregenes encoding proteins excreting L-amino acid from bacterial cell. Suchgenes are exemplified by b2682 and b2683 genes (ygaZH genes) (EP1239041A2).

Examples of bacteria belonging to the genus Escherichia, which have anactivity to produce L-proline include the following E. coli strains:NRRL B-12403 and NRRL B-12404 (GB Patent 2075056), VKPM B-8012 (Russianpatent application No. 2000124295), plasmid mutants described inDE3127361 A1, plasmid mutants described by Bloom F. R. et al. in “The15^(th) Miami winter symposium”, 1983, p. 34, and the like.

L-Threonine-Producing Bacteria

Examples of parental strains which can be used to deriveL-threonine-producing bacteria include, but are not limited to strainsbelonging to the genus Escherichia such as E. coli TDH-6/pVIC40 (VKPMB-3996) (U.S. Pat. No. 5,175,107, U.S. Pat. No. 5,705,371), E. coli472T23/pYN7 (ATCC 98081) (U.S. Pat. No. 5,631,157), E. coli NRRL-21593(U.S. Pat. No. 5,939,307), E. coli FERM BP-3756 (U.S. Pat. No.5,474,918), E. coli FERM BP-3519 and FERM BP-3520 (U.S. Pat. No.5,376,538), E. coli MG442 (Gusyatiner et al., Genetika (Russian), 1978,14:947-956), E. coli VL643 and VL2055 (EP1149911 A2), and the like.

The strain TDH-6 is deficient in the thrC gene, as well as beingsucrose-assimilative, and the ilvA gene has a leaky mutation. Thisstrain also has a mutation in the rhtA gene, which imparts resistance tohigh concentrations of threonine or homoserine. The strain B-3996contains the plasmid pVIC40 which was obtained by inserting a thrA*BCoperon which includes a mutant thrA gene into a RSF1010-derived vector.This mutant thrA gene encodes aspartokinase homoserine dehydrogenase Iwhich has substantially desensitized feedback inhibition by threonine.The strain B-3996 was deposited on Nov. 19, 1987 in the All-UnionScientific Center of Antibiotics (Russian Federation, 117105 Moscow,Nagatinskaya Street 3-A) under the accession number RIA 1867. The strainwas also deposited in the Russian National Collection of IndustrialMicroorganisms (VKPM) (Russian Federation, 117545 Moscow, 1^(st)Dorozhny proezd, 1) on Apr. 7, 1987 under the accession number B-3996.

E. coli VKPM B-5318 (EP0593792 A1) may also be used as a parental strainfor deriving L-threonine-producing bacteria. The strain B-5318 isprototrophic with regard to isoleucine; and a temperature-sensitivelambda-phage C1 repressor and PR promoter replace the regulatory regionof the threonine operon in plasmid pVIC40. The strain VKPM B-5318 wasdeposited in the Russian National Collection of IndustrialMicroorganisms (VKPM) on May 3, 1990 under the accession number of VKPMB-5318.

The bacterium can be additionally modified to enhance expression of oneor more of the following genes:

-   -   the mutant thrA gene which encodes aspartokinase homoserine        dehydrogenase I resistant to feedback inhibition by threonine;    -   the thrB gene which encodes homoserine kinase;    -   the thrC gene which encodes threonine synthase;    -   the rhtA gene which encodes a putative transmembrane protein of        the threonine and homoserine efflux system;    -   the asd gene which encodes aspartate-β-semialdehyde        dehydrogenase; and    -   the aspC gene which encodes aspartate aminotransferase        (aspartate transaminase);

The thrA gene which encodes aspartokinase I and homoserine dehydrogenaseI of E. coli has been elucidated (KEGG entry No. b0002; GenBankaccession No. NC_(—)000913.2; nucleotide positions: 337 to 2,799; GeneID: 945803). The thrA gene is located between the thrL and thrB genes onthe chromosome of E. coli K-12.

The thrB gene which encodes homoserine kinase of E. coli has beenelucidated (KEGG entry No. b0003; GenBank accession No. NC_(—)000913.2;nucleotide positions: 2,801 to 3,733; Gene ID: 947498). The thrB gene islocated between the thrA and thrC genes on the chromosome of E. coliK-12.

The thrC gene which encodes threonine synthase of E. coli has beenelucidated (KEGG entry No. b0004; GenBank accession No. NC_(—)000913.2;nucleotide positions: 3,734 to 5,020; Gene ID: 945198). The thrC gene islocated between the thrB and yaaX genes on the chromosome of E. coliK-12. All three genes function as a single threonine operon thrABC. Toenhance expression of the threonine operon, the attenuator region whichaffects the transcription is desirably removed from the operon(WO2005049808 A1, WO2003097839 A1).

The mutant thrA gene which encodes aspartokinase I and homoserinedehydrogenase I resistant to feedback inhibition by L-threonine, as wellas, the thrB and thrC genes can be obtained as one operon from thewell-known plasmid pVIC40 which is present in the L-threonine-producingE. coli strain VKPM B-3996. Plasmid pVIC40 is described in detail inU.S. Pat. No. 5,705,371.

The rhtA gene which encodes a protein of the threonine and homoserineefflux system (an inner membrane transporter) of E. coli has beenelucidated (KEGG entry No. b0813; GenBank accession No. NC_(—)000913.2;nucleotide positions: 848,433 to 849,320, complement; Gene ID: 947045).The rhtA gene is located between the dps and ompX genes on thechromosome of E. coli K-12 close to the glnHPQ operon, which encodescomponents of the glutamine transport system. The rhtA gene is identicalto the ybiF gene (KEGG entry No. B0813).

The asd gene which encodes aspartate-β-semialdehyde dehydrogenase of E.coli has been elucidated (KEGG entry No. b3433; GenBank accession No.NC_(—)000913.2; nucleotide positions: 3,571,798 to 3,572,901,complement; Gene ID: 947939). The asd gene is located between the glgBand gntU gene on the same strand (yhgN gene on the opposite strand) onthe chromosome of E. coli K-12.

Also, the aspC gene which encodes aspartate aminotransferase of E. colihas been elucidated (KEGG entry No. b0928; GenBank accession No.NC_(—)000913.2; nucleotide positions: 983,742 to 984,932, complement;Gene ID: 945553). The aspC gene is located between the ycbL gene on theopposite strand and the ompF gene on the same strand on the chromosomeof E. coli K-12.

L-Tryptophan-Producing Bacteria

Examples of parental strains which can be used to derive theL-tryptophan-producing bacteria include, but are not limited to strainsbelonging to the genus Escherichia such as E. coli JP4735/pMU3028(DSM10122) and JP6015/pMU91 (DSM10123) deficient in thetryptophanyl-tRNA synthetase encoded by mutant trpS gene (U.S. Pat. No.5,756,345), E. coli SV164 (pGH5) having a serA allele encodingphosphoglycerate dehydrogenase free from feedback inhibition by serineand a trpE allele encoding anthranilate synthase free from feedbackinhibition by tryptophan (U.S. Pat. No. 6,180,373), E. coli AGX17(pGX44) (NRRL B-12263) and AGX6(pGX50)aroP (NRRL B-12264) deficient inthe enzyme tryptophanase (U.S. Pat. No. 4,371,614), E. coli AGX17/pGX50,pACKG4-pps in which a phosphoenolpyruvate-producing ability is enhanced(WO9708333, U.S. Pat. No. 6,319,696), and the like may be used.L-tryptophan-producing bacteria belonging to the genus Escherichia withan enhanced activity of the identified protein encoded by and the yedAgene or the yddG gene may also be used (U.S. patent applications2003/0148473 A1 and 2003/0157667 A1).

Examples of parental strains which can be used to derive theL-tryptophan-producing bacteria also include strains in which one ormore activities of the enzymes selected from anthranilate synthase,phosphoglycerate dehydrogenase, and tryptophan synthase are enhanced.The anthranilate synthase and phosphoglycerate dehydrogenase are bothsubject to feedback inhibition by L-tryptophan and L-serine, so that amutation desensitizing the feedback inhibition may be introduced intothese enzymes. Specific examples of strains having such a mutationinclude an E. coli SV164 which harbors desensitized anthranilatesynthase and a transformant strain obtained by introducing into the E.coli SV164 the plasmid pGH5 (WO 94/08031), which contains a mutant serAgene encoding feedback-desensitized phosphoglycerate dehydrogenase.

Examples of parental strains which can be used to derive theL-tryptophan-producing bacteria also include strains into which thetryptophan operon which contains a gene encoding desensitizedanthranilate synthase has been introduced (JP 57-71397 A, JP 62-244382A, U.S. Pat. No. 4,371,614). Moreover, L-tryptophan-producing abilitymay be imparted by enhancing expression of a gene which encodestryptophan synthase, among tryptophan operons (trpBA). The tryptophansynthase consists of α and β subunits which are encoded by the trpA andtrpB genes, respectively. In addition, L-tryptophan-producing abilitymay be improved by enhancing expression of the isocitrate lyase-malatesynthase operon (WO2005/103275).

L-Valine-Producing Bacteria

Examples of parental strains which can be used to deriveL-valine-producing bacteria include, but are not limited to strainswhich have been modified to overexpress the ilvGMEDA operon (U.S. Pat.No. 5,998,178). It is desirable to remove the region of the ilvGMEDAoperon which is required for attenuation so that expression of theoperon is not attenuated by the L-valine that is produced. Furthermore,the ilvA gene in the operon is desirably disrupted so that threoninedeaminase activity is decreased.

Examples of parental strains for deriving L-valine-producing bacteriaalso include mutants having a mutation of aminoacyl-tRNA synthetase(U.S. Pat. No. 5,658,766). For example, E. coli VL1970, which has amutation in the ileS gene encoding isoleucine tRNA synthetase, can beused. E. coli VL1970 was deposited in the Russian National Collection ofIndustrial Microorganisms (VKPM) (Russian Federation, 117545 Moscow,1^(st) Dorozhny Proezd, 1) on Jun. 24, 1988 under the accession numberVKPM B-4411.

Furthermore, mutants requiring lipoic acid for growth and/or lackingH⁺-ATPase can also be used as parental strains (WO96/06926).

Examples of L-valine-producing strain include E. coli strain H-81 (VKPMB-8066), NRRL B-12287 and NRRL B-12288 (U.S. Pat. No. 4,391,907), VKPMB-4411 (U.S. Pat. No. 5,658,766), VKPM B-7707 (European patentapplication EP1016710 A2), or the like.

The bacterium of the present invention belonging to the familyEnterobacteriaceae has been modified to attenuate expression of the yjjKgene.

The phrase “a bacterium modified to attenuate expression of the yjjKgene” can mean that the bacterium has been modified in such a way thatin the modified bacterium, expression of the yjjK gene is decreased ascompared to a bacterium which contains a non-modified yjjK gene, forexample, a wild-type or parental strain, or the yjjK gene isinactivated.

The phrase “the yjjK gene is inactivated” can mean that the modifiedgene encodes a completely inactive or non-functional protein. It is alsopossible that the modified DNA region is unable to naturally express thegene due to deletion of a part of the gene or deletion of the entiregene, replacement of one base or more to cause an amino acidsubstitution in the protein encoded by the gene (missense mutation),introduction of a stop codon (nonsense mutation), deletion of one or twobases to cause a reading frame shift of the gene, insertion of adrug-resistance gene and/or transcription termination signal, ormodification of an adjacent region of the gene, including sequencescontrolling gene expression such as promoter(s), enhancer(s),attenuator(s), ribosome-binding site(s) (RBS), etc. Inactivation of thegene can also be performed, for example, by conventional methods such asa mutagenesis treatment using UV irradiation or nitrosoguanidine(N-methyl-N′-nitro-N-nitrosoguanidine), site-directed mutagenesis, genedisruption using homologous recombination, and/or insertion-deletionmutagenesis (Yu D. et al., Proc. Natl. Acad. Sci. USA, 2000,97(11):5978-5983; Datsenko K. A. and Wanner B. L., Proc. Natl. Acad.Sci. USA, 2000, 97(12):6640-6645; Zhang Y. et al., Nature Genet., 1998,20:123-128) based on “Red/ET-driven integration” or “λRed/ET-mediatedintegration”.

The phrase “expression of the yjjK gene is attenuated” can mean that anamount of the YjjK protein in the modified bacterium, in whichexpression of the yjjK gene is attenuated, is reduced as compared with anon-modified bacterium, for example, a wild-type or parental strain suchas E. coli K-12.

The phrase “expression of the yjjK gene is attenuated” can also meanthat the modified bacterium contains a region operably linked to thegene, including sequences controlling gene expression such as promoters,enhancers, attenuators and transcription termination signals,ribosome-binding sites (RBS), and other expression control elements,which is modified resulting in a decrease in the expression level of theyjjK gene; and other examples (see, for example, WO95/34672; Carrier T.A. and Keasling J. D., Biotechnol. Prog., 1999, 15:58-64).

Expression of the yjjK gene can be attenuated by replacing an expressioncontrol sequence of the gene, such as a promoter on the chromosomal DNA,with a weaker one. The strength of a promoter is defined by thefrequency of initiation acts of RNA synthesis. Examples of methods forevaluating the strength of promoters and strong promoters are describedin Goldstein et al., Prokaryotic promoters in biotechnology, Biotechnol.Annu. Rev., 1995, 1:105-128), and so forth. Furthermore, it is alsopossible to introduce nucleotide substitution for several nucleotides ina promoter region of a target gene and thereby modify the promoter to beweakened as disclosed in International Patent Publication WO00/18935.Furthermore, it is known that substitution of several nucleotides in theShine-Dalgarno (SD) sequence, and/or in the spacer between the SDsequence and the start codon, and/or a sequence immediately upstreamand/or downstream from the start codon in the ribosome-binding site(RBS) greatly affects the translation efficiency of mRNA. Thismodification of the RBS may be combined with decreasing transcription ofthe yjjK gene.

Expression of the yjjK gene can also be attenuated by insertion of atransposon or an insertion sequence (IS) into the coding region of thegene (U.S. Pat. No. 5,175,107) or in the region controlling geneexpression, or in the proximal part of the yjjK gene structure, wherethe yjjK is the distal part, or by conventional methods such asmutagenesis with ultraviolet irradiation (UV) irradiation ornitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine). Furthermore,the incorporation of a site-specific mutation can be conducted by knownchromosomal editing methods based, for example, on λRed/ET-mediatedrecombination.

The copy number, presence or absence of the gene can be measured, forexample, by restricting the chromosomal DNA followed by Southernblotting using a probe based on the gene sequence, fluorescence in situhybridization (FISH), and the like. The level of gene expression can bedetermined by measuring the amount of mRNA transcribed from the geneusing various well-known methods, including Northern blotting,quantitative RT-PCR, and the like. The amount of the protein encoded bythe gene can be measured by known methods including SDS-PAGE followed byimmunoblotting assay (Western blotting analysis), or mass spectrometryanalysis of the protein samples, and the like.

Methods for manipulation with recombinant molecules of DNA and molecularcloning such as preparation of plasmid DNA, digestion, ligation andtransformation of DNA, selection of an oligonucleotide as a primer,incorporation of mutations, and the like may be ordinary methodswell-known to the person skilled in the art. These methods aredescribed, for example, in Sambrook J., Fritsch E. F. and Maniatis T.,“Molecular Cloning: A Laboratory Manual”, 2^(nd) ed., Cold Spring HarborLaboratory Press (1989) or Green M. R. and Sambrook J. R., “MolecularCloning: A Laboratory Manual”, 4^(th) ed., Cold Spring Harbor LaboratoryPress (2012); Bernard R. Glick, Jack J. Pasternak and Cheryl L. Patten,“Molecular Biotechnology: principles and applications of recombinantDNA”, 4^(th) ed., Washington, DC, ASM Press (2009).

The yjjK gene encodes the fused predicted transporter subunits of ABCsuperfamily, ATP-binding components YjjK (KEGG, Kyoto Encyclopedia ofGenes and Genomes, entry No. b4391; Protein Knowledgebase,UniProtKB/Swiss-Prot, accession No. P0A9W3). The yjjK gene (GenBankaccession No. NC_(—)000913.2; nucleotide positions: 4626878 to 4628545,complement; Gene ID: 948909) is located between the nadR and slt geneson the opposite strand on the chromosome of E. coli strain K-12. Thenucleotide sequence of the yjjK gene and the amino acid sequence of theYjjK protein encoded by the yjjK gene are shown in SEQ ID NO: 1 and SEQID NO: 2, respectively.

The amino acid sequences of the ABC transporter ATP-binding protein YjjKfrom other bacterial species belonging to the family Enterobacteriaceaeare known. The exemplary YjjK proteins are listed, for example, in theProtein Knowledgebase, UniProtKB/Swiss-Prot (www.uniprot.org/) underaccession Nos. F2EU25 (Pantoea ananatis, strain AJ13355, FERM BP-6614),D0ZV61 (Salmonella typhimurium, strain 14028s/SGSC 2262, The SalmonellaGenetic Stock Centre (SGSC), Department of Biological Sciences, 2500University Dr. N. W. Calgary, Alberta, Canada T2N 1N4), P0A9W5 (Shigellaflexneri) or a homolog thereof from Shigella flexneri Castellani andChalmers, ATCC 29903, B2VH30 (Erwinia tasmaniensis, strain DSM17950/Et1/99, Leibniz Institute, DSMZ-German Collection ofMicroorganisms and Cell Cultures, Inhoffenstrasse 7B, 38124Braunschweig, Germany), C7BJV0 (Photorhabdus asymbiotica subsp.asymbiotica, strain ATCC 43949/3105-77), and so forth.

Since there may be some differences in DNA sequences between the genera,species or strains of the family Enterobacteriaceae, the yjjK gene isnot limited to the gene shown in SEQ ID NO: 1, but may include geneswhich are variant nucleotide sequences of or homologous to SEQ ID NO: 1,and which encode variants of the YjjK protein.

The phrase “a variant protein” can mean a protein which has one orseveral changes in the sequence compared with SEQ ID NO: 2, whether theyare substitutions, deletions, insertions, and/or additions of one orseveral amino acid residues, but still maintains an activity or functionsimilar to that of the YjjK protein, or the three-dimensional structureof the YjjK protein is not significantly changed relative to thewild-type or non-modified protein. The number of changes in the variantprotein depends on the position in the three-dimensional structure ofthe protein or the type of amino acid residues. It can be, but is notstrictly limited to, 1 to 30, in another example 1 to 15, in anotherexample 1 to 10, and in another example 1 to 5, in SEQ ID NO: 2. Thesechanges in the variant protein can occur in regions of the protein thatare not critical for the activity or function of the protein. This isbecause some amino acids have high homology to one another so that theactivity or function is not affected by such a change, or thethree-dimensional structure of YjjK is not significantly changedrelative to the wild-type or non-modified protein. Therefore, theprotein variants encoded by the yjjK gene may have a homology, definedas the parameter “identity” when using the computer program BLAST, ofnot less than 80%, not less than 90%, not less than 95%, or not lessthan 98% with respect to the entire amino acid sequence shown in SEQ IDNO: 2, as long as the activity or function of the YjjK protein ismaintained, or the three-dimensional structure of YjjK is notsignificantly changed relative to the wild-type or non-modified protein.

The exemplary substitution, deletion, insertion, and/or addition of oneor several amino acid residues can be a conservative mutation(s). Therepresentative conservative mutation is a conservative substitution. Theconservative substitution can be, but is not limited to a substitution,wherein substitution takes place mutually among Phe, Trp and Tyr, if thesubstitution site is an aromatic amino acid; among Ala, Leu, Ile andVal, if the substitution site is a hydrophobic amino acid; between Glu,Asp, Gln, Asn, Ser, His and Thr, if the substitution site is ahydrophilic amino acid; between Gln and Asn, if the substitution site isa polar amino acid; among Lys, Arg and His, if the substitution site isa basic amino acid; between Asp and Glu, if the substitution site is anacidic amino acid; and between Ser and Thr, if the substitution site isan amino acid having hydroxyl group. Examples of conservativesubstitutions include substitution of Ser or Thr for Ala, substitutionof Gln, His or Lys for Arg, substitution of Glu, Gln, Lys, His or Aspfor Asn, substitution Asn, Glu or Gln for Asp, substitution of Ser orAla for Cys, substitution Asn, Glu, Lys, His, Asp or Arg for Gln,substitution Asn, Gln, Lys or Asp for Glu, substitution of Pro for Gly,substitution Asn, Lys, Gln, Arg or Tyr for His, substitution of Leu,Met, Val or Phe for Ile, substitution of Ile, Met, Val or Phe for Leu,substitution Asn, Glu, Gln, His or Arg for Lys, substitution of Ile,Leu, Val or Phe for Met, substitution of Trp, Tyr, Met, Ile or Leu forPhe, substitution of Thr or Ala for Ser, substitution of Ser or Ala forThr, substitution of Phe or Tyr for Trp, substitution of His, Phe or Trpfor Tyr, and substitution of Met, Ile or Leu for Val.

The exemplary substitution, deletion, insertion, and/or addition of oneor several amino acid residues can also be a non-conservativemutation(s) provided that the mutation(s) is/are compensated by one ormore secondary mutations in the different position(s) of amino acidssequence so that the activity or function of the variant protein ismaintained and similar to that of the YjjK protein, or thethree-dimensional structure of YjjK is not significantly changedrelative to the wild-type or non-modified protein.

To evaluate the degree of protein or DNA homology, several calculationmethods can be used, such as BLAST search, FASTA search and ClustalWmethod. The BLAST (Basic Local Alignment Search Tool,www.ncbi.nlm.nih.gov/BLAST/) search is the heuristic search algorithmemployed by the programs blastp, blastn, blastx, megablast, tblastn, andtblastx; these programs ascribe significance to their findings using thestatistical methods of Samuel K. and Altschul S. F. (“Methods forassessing the statistical significance of molecular sequence features byusing general scoring schemes” Proc. Natl. Acad. Sci. USA, 1990,87:2264-2268; “Applications and statistics for multiple high-scoringsegments in molecular sequences”. Proc. Natl. Acad. Sci. USA, 1993,90:5873-5877). The computer program BLAST calculates three parameters:score, identity and similarity. The FASTA search method is described byPearson W. R. (“Rapid and sensitive sequence comparison with FASTP andFASTA”, Methods Enzymol., 1990, 183:63-98). The ClustalW method isdescribed by Thompson J. D. et al. (“CLUSTAL W: improving thesensitivity of progressive multiple sequence alignment through sequenceweighting, position-specific gap penalties and weight matrix choice”,Nucleic Acids Res., 1994, 22:4673-4680).

Moreover, the yjjK gene can be a variant nucleotide sequence. The phrase“a variant nucleotide sequence” can mean a nucleotide sequence whichencodes the YjjK protein using any synonymous amino acid codonsaccording to the standard genetic code table (see, e.g., Lewin B.,“Genes VIII”, 2004, Pearson Education, Inc., Upper Saddle River, N.J.07458), or “a variant protein” of the YjjK protein. The yjjK gene can bea variant nucleotide sequence due to degeneracy of genetic code.

The phrase “a variant nucleotide sequence” can also mean, but is notlimited to a nucleotide sequence which hybridizes under stringentconditions with the nucleotide sequence complementary to the sequenceshown in SEQ ID NO: 1, or a probe which can be prepared from thenucleotide sequence under stringent conditions provided that it encodesfunctional protein. “Stringent conditions” include those under which aspecific hybrid, for example, a hybrid having homology, defined as theparameter “identity” when using the computer program BLAST, of not lessthan 70%, not less than 80%, not less than 90%, not less than 95%, notless than 96%, not less than 97%, not less than 98%, or not less than99% is formed, and a non-specific hybrid, for example, a hybrid havinghomology lower than the above is not formed. For example, stringentconditions can be exemplified by washing one time or more, or in anotherexample, two or three times, at a salt concentration of 1×SSC (standardsodium citrate or standard sodium chloride), 0.1% SDS (sodium dodecylsulphate), or in another example, 0.1×SSC, 0.1% SDS at 60° C. or 65° C.Duration of washing depends on the type of membrane used for blottingand, as a rule, can be what is recommended by the manufacturer. Forexample, the recommended duration of washing for the Amersham Hybond™-N+positively charged nylon membrane (GE Healthcare) under stringentconditions is 15 minutes. The washing step can be performed 2 to 3times. As the probe, a part of the sequence complementary to thesequences shown in SEQ ID NO: 1 may also be used. Such a probe can beproduced by PCR using oligonucleotides as primers prepared on the basisof the sequence shown in SEQ ID NO: 1 and a DNA fragment containing thenucleotide sequence as a template. The length of the probe isrecommended to be >50 bp; it can be suitably selected depending on thehybridization conditions, and is usually 100 bp to 1 kbp. For example,when a DNA fragment having a length of about 300 bp is used as theprobe, the washing conditions after hybridization can be exemplified by2×SSC, 0.1% SDS at 50° C., 60° C. or 65° C.

As the genes encoding the YjjK proteins of the genera Escherichia,Pantoea, Shigella, Erwinia, and Photorhabdus have already beenelucidated (see above), the variant nucleotide sequences encodingvariant proteins of YjjK proteins can be obtained by PCR (polymerasechain reaction; refer to White T. J. et al., The polymerase chainreaction, Trends Genet., 1989, 5:185-189) utilizing primers preparedbased on the nucleotide sequence of the yjjK gene; or the site-directedmutagenesis method by treating a DNA containing the wild-type or mutantyjjK gene in vitro, for example, with hydroxylamine, or a method fortreating a microorganism, for example, a bacterium belonging to thefamily Enterobacteriaceae harboring the wild-type or mutant yjjK genewith ultraviolet (UV) irradiation or a mutating agent such asN-methyl-N′-nitro-N-nitrosoguanidine (NTG) and nitrous acid usually usedfor the such treatment; or chemically synthesized as full-length genestructure. Genes encoding the YjjK protein or its variant proteins ofother microorganisms can be obtained in a similar manner.

The phrase “a wild-type protein” can mean a native protein naturallyproduced by a wild-type or parent bacterial strain of the familyEnterobacteriaceae, for example, by the wild-type E. coli MG1655 strain.A wild-type protein can be encoded by the wild-type, or non-modified,gene naturally occurring in genome of a wild-type bacterium.

The phrase “operably linked to a gene” can mean that the regulatoryregion(s) is/are linked to the nucleotide sequence of the nucleic acidmolecule or gene of interest in a manner which allows for expression(e.g., enhanced, increased, constitutive, basal, antiterminated,attenuated, deregulated, decreased or repressed expression) of thenucleotide sequence, preferably expression of a gene product encoded bythe nucleotide sequence.

The bacterium as described herein can be obtained by attenuatingexpression of the yjjK gene in a bacterium inherently having an abilityto produce an L-amino acid. Alternatively, the bacterium as describedherein can be obtained by imparting the ability to produce an L-aminoacid to a bacterium already having attenuated expression of the yjjKgene.

The bacterium can have, in addition to the properties already mentioned,other specific properties such as various nutrient requirements, drugresistance, drug sensitivity, and drug dependence, without departingfrom the scope of the present invention.

2. Method

A method of the present invention includes the method for producing anL-amino acid such as L-alanine, L-arginine, L-asparagine, L-asparticacid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine,L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine,L-tryptophan, L-tyrosine, and L-valine, or a salt thereof, or a mixturethereof. The method for producing an L-amino acid can include the stepsof cultivating the bacterium in a culture medium to allow the L-aminoacid to be produced, excreted, or accumulated in the culture medium, andcollecting the L-amino acid from the culture medium and/or the bacterialcells. Collected amino acid can be further purified. The L-amino acidcan be produced in a salt form thereof. For example, sodium, potassium,ammonium, and the like salts of the L-amino acid can be produced by themethod.

The cultivation of the bacterium, and collection and purification ofL-amino acid or a salt thereof from the medium and the like may beperformed in a manner similar to conventional fermentation methodswherein L-amino acid is produced using a microorganism. The culturemedium for production of the L-amino acid can be either a synthetic ornatural medium such as a typical medium that contains a carbon source, anitrogen source, a sulphur source, inorganic ions, and other organic andinorganic components as required. As the carbon source, saccharides suchas glucose, lactose, galactose, fructose, sucrose, arabinose, maltose,xylose, trehalose, ribose, and hydrolyzates of starches; alcohols suchas glycerol, mannitol, and sorbitol; organic acids such as gluconicacid, fumaric acid, citric acid, malic acid, and succinic acid; and thelike can be used. As the nitrogen source, inorganic ammonium salts suchas ammonium sulfate, ammonium chloride, and ammonium phosphate; organicnitrogen such as of soy bean hydrolyzates; ammonia gas; aqueous ammonia;and the like can be used. The sulphur source can include ammoniumsulphate, magnesium sulphate, ferrous sulphate, manganese sulphate, andthe like. Vitamins such as vitamin B1, required substances, for example,organic nutrients such as nucleic acids such as adenine and RNA, oryeast extract, and the like may be present in appropriate, even iftrace, amounts. Other than these, small amounts of calcium phosphate,iron ions, manganese ions, and the like may be added, if necessary.

Cultivation can be performed under aerobic conditions for 16 to 72 h, orfor 16 to 65 h; the culture temperature during cultivation can becontrolled within 30 to 45° C., or within 30 to 37° C.; and the pH canbe adjusted between 5 and 8, or between 6 and 7.5. The pH can beadjusted by using an inorganic or organic acidic or alkaline substance,as well as ammonia gas.

After cultivation, solids such as cells and cell debris can be removedfrom the liquid medium by centrifugation or membrane filtration, andthen the target L-amino acid or a salt thereof can be recovered from thefermentation liquor by any combination of conventional techniques suchas concentration, ion-exchange chromatography, and crystallization.

The collected target L-amino acid may contain microbial cells, mediumcomponents, moisture, and by-product metabolites of the microorganism inaddition to the target substance. Purity of the collected targetsubstance is 50% or higher, 85% or higher, or 95% or higher (U.S. Pat.No. 5,431,933, Japanese Patent No. 1214636, U.S. Pat. Nos. 4,956,471,4,777,051, 4,946,654, 5,840,358, 6,238,714, U.S. Patent PublishedApplication No. 2005/0025878).

EXAMPLES

The present invention will be more precisely explained below withreference to the following non-limiting Examples.

Example 1 Construction of the E. coli Strain in which the yjjK Gene isInactivated

The yjjK gene was deleted using the method developed by Datsenko K. A.and Wanner B. L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645)called “λRed/ET-mediated integration”. According to this method, the PCRprimers P1 (SEQ ID NO: 3) and P2 (SEQ ID NO: 4), which are homologous toregions adjacent to the yjjK gene at either end, and the gene conferringkanamycin resistance (Km^(R)) in the template chromosome, wereconstructed. The chromosome of E. coli MG1655 ΔattBphi80 nativeIS5.11::LattBphi80-Km^(R)-RattBphi80 (Minaeva N. I. et al., Dual-In/Outstrategy for genes integration into bacterial chromosome: a novelapproach to step-by-step construction of plasmid-less marker-lessrecombinant E. coli strains with predesigned genome structure. BMCBiotechnol., 2008, 8:63) was used as the template. Conditions for PCRwere as follows: initial denaturation for 3 min at 95° C.; profile fortwo first cycles: 1 min at 95° C., 30 sec at 34° C., 40 sec at 72° C.;profile for the last 30 cycles: 30 sec at 95° C., 30 sec at 50° C., 40sec at 72° C.; final step: 5 min at 72° C.

The obtained PCR product 1 (SEQ ID NO: 5) (1,922 bp) was purified by theagarose gel electrophoresis and used for electroporation of the strainE. coli MG1655 ΔattBphi80 native (Minaeva N. I. et al., BMC Biotechnol.,2008, 8:63) containing the plasmid pKD46 with a temperature-sensitivereplication origin. The plasmid pKD46 (Datsenko K. A. and Wanner B. L.,Proc. Natl. Acad. Sci. USA, 2000, 97:12:6640-45) includes the 2,154nucleotides (31088-33241) DNA-fragment of phage λ (GenBank accession No.J02459) and contains genes of the λRed/ET-mediated integration system(γ, β, exo genes) under the control of arabinose-inducible promoterP_(araB). The plasmid pKD46 is necessary to integrate the PCR-productinto the chromosome of strain E. coli MG1655ΔattBphi80 native.

Electrocompetent cells were prepared as follows: E. coli MG1655ΔattBphi80 native was grown overnight at 30° C. in LB-medium containingampicillin (100 mg/L), and the culture was diluted 100 times with 5 mLof SOB-medium (Sambrook J., Fritsch E. F. and Maniatis T., “MolecularCloning: A Laboratory Manual”, 2^(nd) ed., Cold Spring Harbor LaboratoryPress (1989)) containing ampicillin and L-arabinose (1 mM). The obtainedculture was grown with aeration at 30° C. to OD₆₀₀ of ˜0.6 and then madeelectrocompetent by concentrating 100-fold and washing three times withice-cold deionized H₂O. Electroporation was performed using 70 μL ofcells and ˜100 ng of the PCR product 1. Cells were incubated in 1 mL ofSOC-medium (Sambrook J., Fritsch E. F. and Maniatis T., “MolecularCloning: A Laboratory Manual”, 2^(nd) ed., Cold Spring Harbor LaboratoryPress (1989)) at 37° C. for 2.5 h, plated onto plates containing thelysogenic broth (Sambrook, J. and Russell, D. W. “Molecular Cloning: ALaboratory Manual”, 3^(rd) ed., Cold Spring Harbor Laboratory Press(2001)), agar (1.5%) and kanamycin (50 mg/L), and grown at 37° C. toselect Km^(R) recombinants. Two passages on L-agar with kanamycin (50mg/L) at 42° C. were performed to eliminate the pKD46 plasmid, and thecolonies obtained were tested for sensitivity to ampicillin usingstandard procedure. Thus the E. coli MG1655 ΔattBphi80 nativeΔyjjK::Km^(R) strain was obtained.

Verification of the yjjK Gene Deletion by PCR

Mutants containing deletion of the yjjK gene and marked withkanamycin-resistance gene (kan) were verified by PCR. Locus-specificprimers P3 (SEQ ID NO: 6) and P4 (SEQ ID NO: 7) were used for PCR.Conditions were as follows: initial denaturation for 3 min at 94° C.;profile for the 30 cycles: 30 sec at 94° C., 30 sec at 53° C., 2 min at72° C.; final step: 6 min at 72° C. The PCR product 2 (SEQ ID NO: 8)(1,783 bp) was obtained, when the chromosomal DNA from parental yjjKstrain E. coli MG1655 ΔattBphi80 native was used as the template. ThePCR product 3 (SEQ ID NO: 9) (1,989 bp) was obtained, when thechromosomal DNA from mutant MG1655 ΔattBphi80 native ΔyjjK::Km^(R)strain was used as the template.

Example 2 Construction of the E. coli L-Valine-Producing Strain in whichthe yjjK Gene is Inactivated

The yjjK gene was deleted from the E. coli H-81 L-valine-producingstrain using the P1-transduction (Miller J. H. “Experiments in moleculargenetics”, Cold Spring Harbor Laboratory, Cold Spring Harbor (1972)).The strain H-81 (EP1239041 A2) was deposited in the Russian NationalCollection of Industrial Microorganisms (VKPM) (Russian Federation,117545 Moscow, 1^(st) Dorozhny proezd, 1) on Jan. 30, 2001 under theaccession number VKPM B-8066, and it was then converted to aninternational deposit under the provisions of the Budapest Treaty onFeb. 1, 2002. The E. coli MG1655 ΔattBphi80 native ΔyjjK::Km^(R) strain(Example 1) was used as the donor. The yjjK-deficient mutants of E. coliH-81 were selected on the plates containing the lysogenic broth(Sambrook, J. and Russell, D. W. “Molecular Cloning: A LaboratoryManual”, 3^(rd) ed., Cold Spring Harbor Laboratory Press (2001)), agar(1.5%) and kanamycin (50 mg/L). Thus the E. coli H-81ΔyjjK::Km^(R)strain was obtained. The ΔyjjK::Km^(R) deletion was verified by PCR asdescribed in Example 1.

Example 3 Production of L-Valine by the E. coli H-81ΔyjjK::Km^(R) Strain

The modified E. coli H-81ΔyjjK::Km^(R) and the control E. coli H-81strains were each cultivated at 32° C. for 18 hours in Luria-Bertanibroth (also referred to as lysogenic broth as described in Sambrook, J.and Russell, D. W. “Molecular Cloning: A Laboratory Manual”, 3^(rd) ed.,Cold Spring Harbor Laboratory Press (2001)). Then, 0.2 mL of theobtained culture was inoculated into 2 mL of a fermentation medium in20×200-mm test tubes and cultivated at 30° C. for 48 hours on a rotaryshaker at 250 rpm.

The composition of the fermentation medium (g/L) was as follows:

Glucose 60.0 (NH₄)₂SO₄ 15.0 KH₂PO₄ 1.5 MgSO₄•7H₂O 1.0 Thiamine-HCl 0.1CaCO₃ 25.0 LB medium 10% (v/v) The fermentation medium was sterilized at116° C. for 30 min, except that glucose and CaCO₃ were sterilizedseparately as follows: glucose at 110° C. for 30 min and CaCO₃ at 116°C. for 30 min. The pH was adjusted to 7.0 by KOH solution.

After cultivation, accumulated L-valine was measured using thin-layerchromatography (TLC). TLC plates (10×20 cm) were coated with 0.11-mmlayers of Sorbfil silica gel containing non-fluorescent indicator(Sorbpolymer, Krasnodar, Russian Federation). Samples were applied tothe plates with the Camag Linomat 5 sample applicator. The Sorbfilplates were developed with a mobile phase consisting ofiso-propanol:ethylacetate:25% aqueous ammonia:water (16:16:5:10, v/v). Asolution of ninhydrin (2%, w/v) in acetone was used as the visualizingreagent. After development, plates were dried and scanned with the CamagTLC Scanner 3 in absorbance mode with detection at 520 nm using winCATSsoftware (version 1.4.2).

The results of 9 independent test-tube fermentations are shown inTable 1. As it can be seen from the Table 1, the modified E. coliH-81ΔyjjK::Km^(R) strain was able to produce a higher amount of L-valine(Val) as compared with the parent E. coli H-81 strain.

TABLE 1 Production of L-valine. Strain OD₅₅₀ Val, g/L E. coli H-81(control) 23 ± 1 8.7 ± 0.6 E. coli H-81ΔyjjK::Km^(R) 24 ± 1 9.4 ± 0.1

Example 4 Construction of the E. coli L-Histidine-Producing Strain inwhich the yjjK Gene is Inactivated

To test the effect of inactivation of the yjjK gene on L-histidineproduction, the DNA-fragments from the chromosome of the above-describedE. coli MG1655 ΔattBphi80 native ΔyjjK::Km^(R) strain (Example 1) weretransferred to the L-histidine-producing strain E. coli MG1655+hisGrhisL′_Δ ΔpurR using the P1-transduction (Miller J. H. “Experiments inmolecular genetics”, Cold Spring Harbor Laboratory, Cold Spring Harbor(1972)) as described in Example 2. The ΔyjjK::Km^(R) deletion wasverified by PCR. Thus the E. coli MG1655+hisGr hisL′_Δ ΔpurRΔyjjK::Km^(R) strain was obtained.

The strain E. coli MG1655+hisGr hisL′_Δ ΔpurR has been described inRU2119536 C1; Doroshenko V. G. et al., The directed modification ofEscherichia coli MG1655 to obtain histidine-producing mutants, Prikl.Biochim. Mikrobiol. (Russian), 2013, 49(2):149-154.

Example 5 Production of L-Histidine by the E. coli MG1655+hisGr hisL′_ΔΔpurR Strain

The modified E. coli MG1655+hisGr hisL′_Δ ΔpurR ΔyjjK::Km^(R) and thecontrol E. coli MG1655+hisGr hisL′_Δ ΔpurR strains were each cultivatedat 30° C. for 3 hours in 2 mL L-broth (Sambrook, J. and Russell, D. W.“Molecular Cloning: A Laboratory Manual”, 3^(rd) ed., Cold Spring HarborLaboratory Press (2001)). Then, 0.1 mL of the obtained cultures wereinoculated into 2 mL of fermentation medium in 20×200-mm test tubes andcultivated for 65 h at 30° C. on a rotary shaker (250 rpm).

The composition of the fermentation medium (g/L) was as follows:

Glucose 50.0 Mameno* 0.2 (as the amount of nitrogen) L-aspartate 1.0(NH₄)₂SO₄ 18.0 KCl 1.0 KH₂PO₄ 0.5 MgSO₄•7H₂0 0.4 FeSO₄•7H₂0 0.02MnSO₄•5H₂O 0.02 ZnSO₄•7H₂0 0.02 Adenosine 0.2 Thiamine-HCl 0.001 Betaine2.0 CaCO₃ 60.0 *Mameno is the soybean meal hydrolysate (Ajinomoto Co,Inc.). Glucose, magnesium sulphate, betaine, and CaCO₃ were sterilizedseparately. The pH was adjusted to 6.0 by 6M KOH solution beforesterilization.

After cultivation, accumulated L-histidine was measured using thin-layerchromatography (TLC). TLC plates (10×20 cm) were coated with 0.11-mmlayers of Sorbfil silica gel containing non-fluorescent indicator(Sorbpolymer, Krasnodar, Russian Federation). Samples were applied tothe plates with the Camag Linomat 5 sample applicator. The Sorbfilplates were developed with a mobile phase consisting ofiso-propanol:acetone:25% aqueous ammonia:water (6:6:1.5:1, v/v). Asolution of ninhydrin (2%, w/v) in acetone was used as the visualizingreagent. After development, plates were dried and scanned with the CamagTLC Scanner 3 in absorbance mode with detection at 520 nm using winCATSsoftware (version 1.4.2).

The results of 7 independent test-tube fermentations are shown in Table2. As it can be seen from the Table 2, the modified E. coli MG1655+hisGrhisL′_Δ ΔpurR ΔyjjK::Km^(R) strain was able to produce a higher amountof L-histidine (His) as compared with the parent E. coli MG1655+hisGrhisL′_Δ ΔpurR strain.

TABLE 2 Production of L-histidine. Strain OD₅₅₀ His, g/L E. coliMG1655 + hisGr hisL′_Δ ΔpurR 18.3 ± 0.5 0.90 ± 0.05 (control) E. coliMG1655 + hisGr hisL′_Δ ΔpurR 19.1 ± 1.0 1.10 ± 0.10 ΔyjjK::Km^(R)

Example 6 Production of L-Arginine by E. coli 382ΔyjjK Strain

To test the effect of inactivation of the yjjK gene on L-arginineproduction, the DNA-fragments from the chromosome of the above-describedE. coli MG1655 ΔattBphi80 native ΔyjjK::Km^(R) are transferred to thearginine-producing E. coli strain 382 by P1-transduction (Miller, J. H.“Experiments in Molecular Genetics”, Cold Spring Harbor Lab. Press,Plainview, N.Y. (1972)) to obtain the strain 382ΔyjjK. The strain 382was deposited in the Russian National Collection of IndustrialMicroorganisms (VKPM) (Russian Federation, 117545 Moscow, 1^(st)Dorozhny proezd, 1) on Apr. 10, 2000 under the accession number VKPMB-7926 and then converted to a deposit under the Budapest Treaty on May18, 2001.

Both E. coli strains, 382 and 382ΔyjjK, are separately cultivated withshaking (220 rpm) at 37° C. for 18 h in 3 mL of nutrient broth, and 0.3mL of the obtained cultures are inoculated into 2 mL of a fermentationmedium in 20×200-mm test tubes and cultivated at 32° C. for 48 h on arotary shaker (220 rpm).

After the cultivation, the amount of L-arginine which accumulates in themedium is determined by paper chromatography using a mobile phaseconsisting of n-butanol:acetic acid:water=4:1:1 (v/v). A solution ofninhydrin (2%) in acetone is used as a visualizing reagent. A spotcontaining L-arginine is cut out, L-arginine is eluted with 0.5% watersolution of CdCl₂, and the amount of L-arginine is estimatedspectrophotometrically at 540 nm.

The composition of the fermentation medium (g/L) is as follows:

Glucose 48.0 (NH₄)₂SO₄ 35.0 KH₂PO₄ 2.0 MgSO₄•7H₂O 1.0 Thiamine-HCl0.0002 Yeast extract 1.0 L-isoleucine 0.1 CaCO₃ 5.0 Glucose andmagnesium sulfate are sterilized separately. CaCO₃ is dry-heatsterilized at 180° C. for 2 h. The pH is adjusted to 7.0.

Example 7 Production of L-Cysteine by E. coli JM15(ydeD)ΔyjjK

To test the effect of inactivation of the yjjK gene on L-cysteineproduction, the DNA fragments from the chromosome of the above-describedE. coli MG1655 ΔattBphi80 native ΔyjjK::Km^(R) are transferred to the E.coli L-cysteine-producing strain JM15(ydeD) by P1-transduction to obtainthe strain JM15(ydeD)ΔyjjK.

E. coli JM15(ydeD) is a derivative of E. coli JM15 (U.S. Pat. No.6,218,168), which is transformed with DNA having the ydeD gene encodinga membrane protein, and is not involved in a biosynthetic pathway of anyL-amino acid (U.S. Pat. No. 5,972,663).

Fermentation conditions and procedure for evaluation of L-cysteineproduction were described in detail in Example 6 of U.S. Pat. No.6,218,168.

Example 8 Production of L-Glutamic Acid by E. coli VL334thrC⁺ΔyjjK

To test the effect of inactivation of the yjjK gene on L-glutamic acidproduction, the DNA fragments from the chromosome of the above-describedE. coli MG1655 ΔattBphi80 native ΔyjjK::Km^(R) are transferred to the E.coli L-glutamate-producing strain VL334thrC⁺ (EP1172433 A1) byP1-transduction to obtain the strain VL334thrC⁺ΔyjjK. The strainVL334thrC⁺ has been deposited in the Russian National Collection ofIndustrial Microorganisms (VKPM) (Russian Federation, 117545 Moscow, 1Dorozhny proezd, 1) on Dec. 6, 2004 under the accession number B-8961and then converted to a deposit under the Budapest Treaty on Dec. 8,2004.

E. coli strains, VL334thrC⁺ and VL334thrC⁺ ΔyjjK, are separatelycultivated for 18-24 h at 37° C. on L-agar plates. Then, one loop of thecells is transferred into test tubes containing 2 mL of fermentationmedium.

The composition of the fermentation medium (g/L) is as follows:

Glucose 60.0 (NH₄)₂SO₄ 25.0 KH₂PO₄ 2.0 MgSO₄•7H₂O 1.0 Thiamine-HCl 0.1L-isoleucine 0.07 CaCO₃ 25.0 Glucose and CaCO₃ are sterilizedseparately. The pH is adjusted to 7.2.

Cultivation is carried out at 30° C. for 3 days with shaking. After thecultivation, the amount of L-glutamic acid, which is produced, isdetermined by paper chromatography using a mobile phase consisting ofn-butanol:acetic acid:water=4:1:1 with subsequent staining by ninhydrin(1% solution in acetone), elution of the compounds in 50% ethanol with0.5% CdCl₂ and further estimation of L-glutamic acid at 540 nm.

Example 9 Production of L-Leucine by E. coli 57ΔyjjK

To test the effect of inactivation of the yjjK gene on L-leucineproduction, the DNA fragments from the chromosome of the above-describedE. coli MG1655 ΔattBphi80 native ΔyjjK::Km^(R) are transferred to the E.coli L-leucine-producing strain 57 (VKPM B-7386, U.S. Pat. No.6,124,121) by P1-transduction to obtain the strain 57ΔyjjK. The strain57 has been deposited in the Russian National Collection of IndustrialMicroorganisms (VKPM) (Russian Federation, 117545 Moscow, 1^(st)Dorozhny proezd, 1) on May 19, 1997 under the accession number B-7386.

E. coli strains, 57 and 57ΔyjjK, are separately cultivated for 18-24 hat 37° C. on L-agar plates. To obtain a seed culture, the strains aregrown on a rotary shaker (250 rpm) at 32° C. for 18 h in 20×200-mm testtubes containing 2 mL of L-broth (Sambrook, J. and Russell, D. W. (2001)“Molecular Cloning: A Laboratory Manual”, 3^(rd) ed., Cold Spring HarborLaboratory Press) supplemented with sucrose (4%). Then, the fermentationmedium is inoculated with 0.2 mL of seed material (10%). Thefermentation is performed in 2 mL of a minimal fermentation medium in20×200-mm test tubes. Cells are grown for 48-72 h at 32° C. with shakingat 250 rpm. The amount of L-leucine which accumulates in the medium ismeasured by paper chromatography using a mobile phase consisting ofn-butanol:acetic acid:water=4:1:1.

The composition of the fermentation medium (g/L) is as follows:

Glucose 60.0 (NH₄)₂SO₄ 25.0 K₂HPO₄ 2.0 MgSO₄•7H₂O 1.0 Thiamine-HCl 0.01CaCO₃ 25.0 Glucose is sterilized separately. CaCO₃ is dry-heatsterilized at 180° C. for 2 h. The pH is adjusted to 7.2.

Example 10 Production of L-Lysine by E. coli AJ11442ΔyjjK

To test the effect of inactivation of the yjjK gene on L-lysineproduction, the DNA fragments from the chromosome of the above-describedE. coli MG1655 ΔattBphi80 native ΔyjjK::Km^(R) are transferred to theL-lysine-producing E. coli strain AJ11442 by P1-transduction to obtainthe AJ11442ΔyjjK strain. The strain AJ11442 was deposited inFermentation Research Institute, Agency of Industrial Science andTechnology (currently, Incorporated Administrative Agency, NationalInstitute of Technology and Evaluation, International Patent Organism(NITE IPOD), Kazusakamatari, Kisarazu-shi, Chiba-ken 292-0818, JAPAN) onMay 5, 1981 under a deposition number of FERM P-5084, and transferredfrom the original deposition to international deposition based onBudapest Treaty on Oct. 29, 1987, and has been deposited as depositionnumber of FERM BP-1543. The pCABD2 plasmid includes the dapA geneencoding dihydrodipicolinate synthase having a mutation whichdesensitizes feedback inhibition by L-lysine, the lysC gene encodingaspartokinase III having a mutation which desensitizes feedbackinhibition by L-lysine, the dapB gene encoding dihydrodipicolinatereductase, and the ddh gene encoding diaminopimelate dehydrogenase (U.S.Pat. No. 6,040,160).

E. coli strains, AJ11442 and AJ11442ΔyjjK, are separately cultivated inL-medium containing streptomycin (20 mg/L) at 37° C., and 0.3 mL of theobtained culture is inoculated into 20 mL of the fermentation mediumcontaining the required drugs in a 500-mL flask. The cultivation iscarried out at 37° C. for 16 h by using a reciprocal shaker at theagitation speed of 115 rpm. After the cultivation, the amounts ofL-lysine and residual glucose in the medium are measured by a knownmethod (Biotech-analyzer AS210, Sakura Seiki Co.). Then, the yield ofL-lysine is calculated relative to consumed glucose for each of thestrains.

The composition of the fermentation medium (g/L) is as follows:

Glucose 40.0 (NH₄)₂SO₄ 24.0 K₂HPO₄ 1.0 MgSO₄•7H₂O 1.0 FeSO₄•7H₂O 0.01MnSO₄•5H₂O 0.01 Yeast extract 2.0 The pH is adjusted to 7.0 by KOH andthe medium is autoclaved at 115° C. for 10 min. Glucose and magnesiumsulfate are sterilized separately. CaCO₃ is dry-heat sterilized at 180°C. for 2 h and added to the medium for a final concentration of 30 g/L.

Example 11 Production of L-Phenylalanine by E. coli AJ12739ΔyjjK

To test the effect of inactivation of the yjjK gene on L-phenylalanineproduction, the DNA fragments from the chromosome of the above-describedE. coli MG1655 ΔattBphi80 native ΔyjjK::Km^(R) are transferred to thephenylalanine-producing E. coli strain AJ12739 by P1-transduction toobtain strain AJ12739ΔyjjK. The strain AJ12739 has been deposited in theRussian National Collection of Industrial Microorganisms (VKPM) (RussianFederation, 117545 Moscow, 1^(st) Dorozhny proezd, 1) on Nov. 6, 2001under the accession number VKPM B-8197 and then converted to a depositunder the Budapest Treaty on Aug. 23, 2002.

E. coli strains, AJ12739 and AJ12739ΔyjjK, are separately cultivated at37° C. for 18 h in a nutrient broth, and 0.3 mL of the obtained cultureis each inoculated into 3 mL of a fermentation medium in 20×200-mm testtubes and cultivated at 37° C. for 48 h with shaking on a rotary shaker.After cultivation, the amount of L-phenylalanine which accumulates inthe medium is determined by thin layer chromatography (TLC). The10×15-cm TLC plates coated with 0.11-mm layers of Sorbfil silica gelcontaining non-fluorescent indicator (Stock Company Sorbpolymer,Krasnodar, Russian Federation) are used. The Sorbfil plates aredeveloped with a mobile phase consisting of propan-2-ol:ethylacetate:25%aqueous ammonia:water=40:40:7:16 (v/v). A solution of ninhydrin (2%) inacetone is used as a visualizing reagent.

The composition of the fermentation medium (g/L) is as follows:

Glucose 40.0 (NH₄)₂SO₄ 16.0 K₂HPO₄ 0.1 MgSO₄•7H₂O 1.0 FeSO₄•7H₂O 0.01MnSO₄•5H₂O 0.01 Thiamine-HCl 0.0002 Yeast extract 2.0 Tyrosine 0.125CaCO₃ 20.0 Glucose and magnesium sulfate are sterilized separately.CaCO₃ is dry-heat sterilized at 180° C. for 2 h. The pH is adjusted to7.0.

Example 12 Production of L-Proline by E. coli 702ilvAΔyjjK

To test the effect of inactivation of the yjjK gene on L-prolineproduction, the DNA fragments from the chromosome of the above-describedE. coli MG1655 ΔattBphi80 native ΔyjjK::Km^(R) are transferred to theproline-producing E. coli strain 702ilvA by P1-transduction to obtainthe strain 702ilvAΔyjjK. The strain 702ilvA was deposited in the RussianNational Collection of Industrial Microorganisms (VKPM) (RussianFederation, 117545 Moscow, 1^(st) Dorozhny proezd, 1) on Jul. 18, 2000under the accession number VKPM B-8012 and was then converted to adeposit under the Budapest Treaty on May 18, 2001.

E. coli strains, 702ilvA and 702ilvAΔyjjK, are separately cultivated for18-24 h at 37° C. on L-agar plates. Then, these strains are cultivatedunder the same conditions as in Example 8 (Production of L-glutamicacid).

Example 13 Production of L-Threonine by E. coli B-3996ΔyjjK

To test the effect of inactivation of the yjjK gene on L-threonineproduction, the DNA fragments from the chromosome of the above-describedE. coli MG1655 ΔattBphi80 native ΔyjjK::Km^(R) are transferred to theL-threonine-producing E. coli strain VKPM B-3996 by P1-transduction toobtain the strain B-3996ΔyjjK. The strain B-3996 was deposited on Nov.19, 1987 in the All-Union Scientific Center of Antibiotics (RussianFederation, 117105 Moscow, Nagatinskaya Street, 3-A) under the accessionnumber RIA 1867. The strain was also deposited in the Russian NationalCollection of Industrial Microorganisms (VKPM) (Russian Federation,117545 Moscow, 1^(st) Dorozhny proezd, 1) under the accession numberB-3996.

Both E. coli strains, B-3996 and B-3996ΔyjjK, are separately cultivatedfor 18-24 h at 37° C. on L-agar plates. To obtain a seed culture, thestrains are grown on a rotary shaker (250 rpm) at 32° C. for 18 h in20×200-mm test tubes containing 2 mL of L-broth (Sambrook, J. andRussell, D. W. (2001) “Molecular Cloning: A Laboratory Manual”, 3^(rd)ed., Cold Spring Harbor Laboratory Press) supplemented with glucose(4%). Then, the fermentation medium is inoculated with 0.2 mL (10%) ofseed material. The fermentation is performed in 2 mL of minimal mediumin 20×200-mm test tubes. Cells are grown for 65 h at 32° C. with shakingat 250 rpm.

After cultivation, the amount of L-threonine which accumulates in themedium is determined by paper chromatography using a mobile phaseconsisting of n-butanol:acetic acid:water=4:1:1 (v/v). A solution ofninhydrin (2%) in acetone is used as a visualizing reagent. A spotcontaining L-threonine is cut out, L-threonine is eluted with 0.5% watersolution of CdCl₂, and the amount of L-threonine is estimatedspectrophotometrically at 540 nm.

The composition of the fermentation medium (g/L) is as follows:

Glucose 80.0 (NH₄)₂SO₄ 22.0 NaCl 0.8 KH₂PO₄ 2.0 MgSO₄•7H₂O 0.8FeSO₄•7H₂O 0.02 MnSO₄•5H₂O 0.02 Thiamine-HCl 0.0002 Yeast extract 1.0CaCO₃ 30.0 Glucose and magnesium sulfate are sterilized separately.CaCO₃ is sterilized by dry-heat at 180° C. for 2 h. The pH is adjustedto 7.0. The antibiotic is introduced into the medium aftersterilization.

Example 14 Production of L-Tryptophan by E. coli SV164(pGH5)ΔyjjK

To test the effect of inactivation of the yjjK gene on L-tryptophanproduction, the DNA fragments from the chromosome of the above-describedE. coli MG1655 ΔattBphi80 native ΔyjjK::Km^(R) are transferred to theL-tryptophan-producing E. coli strain SV164(pGH5) by P1-transduction toobtain the strain SV164(pGH5)ΔyjjK. The strain SV164 has the trpE alleleencoding anthranilate synthase free from feedback inhibition bytryptophan. The plasmid pGH5 harbors a mutant serA gene encodingphosphoglycerate dehydrogenase free from feedback inhibition by serine.The strain SV164(pGH5) was described in detail in U.S. Pat. No.6,180,373 or EP0662143 B1.

E. coli strains, SV164(pGH5) and SV164(pGH5)ΔyjjK, are separatelycultivated with shaking at 37° C. for 18 h in 3 mL of nutrient brothsupplemented with tetracycline (20 mg/L, marker of pGH5 plasmid). Theobtained cultures (0.3 mL each) are inoculated into 3 mL of afermentation medium containing tetracycline (20 mg/L) in 20×200-mm testtubes, and cultivated at 37° C. for 48 h with a rotary shaker at 250rpm. After cultivation, the amount of L-tryptophan which accumulates inthe medium is determined by TLC as described in Example 11 (Productionof L-phenylalanine). The fermentation medium components are listed inTable 3, but should be sterilized in separate groups (A, B, C, D, E, F,and H), as shown, to avoid adverse interactions during sterilization.

TABLE 3 Final concentration, Solutions Component g/L A KH₂PO₄ 1.5 NaCl0.5 (NH₄)₂SO₄ 1.5 L-Methionine 0.05 L-Phenylalanine 0.1 L-Tyrosine 0.1Mameno* (as the amount of nitrogen) 0.07 B Glucose 40.0 MgSO₄•7H₂O 0.3 CCaCl₂ 0.011 D FeSO₄•7H₂O 0.075 Sodium citrate 1.0 E Na₂MoO₄•2H₂O 0.00015H₃BO₃ 0.0025 CoCl₂•6H₂O 0.00007 CuSO₄•5H₂O 0.00025 MnCl₂•4H₂O 0.0016ZnSO₄•7H₂O 0.0003 F Thiamine-HCl 0.005 G CaCO₃ 30.0 H Pyridoxine 0.03The pH of solution A is adjusted to 7.1 with NH₄OH. *Mameno is thesoybean meal hydrolysate (Ajinomoto Co, Inc.).

Example 15 Production of L-Citrulline by E. coli 382ΔargGΔyjjK

To test the effect of inactivation of the yjjK gene on L-citrullineproduction, the DNA fragments from the chromosome of the above-describedE. coli MG1655 ΔattBphi80 native ΔyjjK::Km^(R) are transferred to theL-citrulline producing E. coli strain 382ΔargG by P1-transduction toobtain the strain 382ΔargGΔyjjK. The strain 382ΔargG is obtained bydeletion of argG gene on the chromosome of 382 strain (VKPM B-7926) bythe method initially developed by Datsenko K. A. and Wanner B. L. called“λRed/ET-mediated integration” (Datsenko K. A. and Wanner B. L., Proc.Natl. Acad. Sci. USA, 2000, 97(12):6640-6645). According to thisprocedure, the PCR primers homologous to both the region adjacent to theargG gene and the gene which confers antibiotic resistance in thetemplate plasmid are constructed. The plasmid pMW118-λattL-cat-λattR (WO05/010175) is used as the template in the PCR reaction.

Both E. coli strains, 382ΔargG and 382ΔargGΔyjjK, are separatelycultivated with shaking at 37° C. for 18 h in 3 mL of nutrient broth,and 0.3 mL of the obtained cultures are inoculated into 2 mL of afermentation medium in 20×200-mm test tubes and cultivated at 32° C. for48 h on a rotary shaker.

After the cultivation, the amount of L-citrulline which accumulates inthe medium is determined by paper chromatography using a mobile phaseconsisting of n-butanol:acetic acid:water=4:1:1 (v/v). A solution ofninhydrin (2%) in acetone is used as a visualizing reagent. A spotcontaining citrulline is cut out, L-citrulline is eluted with 0.5% watersolution of CdCl₂, and the amount of L-citrulline is estimatedspectrophotometrically at 540 nm.

The composition of the fermentation medium (g/L) is as follows:

Glucose 48.0 (NH₄)₂SO₄ 35.0 KH₂PO₄ 2.0 MgSO₄•7H₂O 1.0 Thiamine-HCl0.0002 Yeast extract 1.0 L-isoleucine 0.1 L-arginine 0.1 CaCO₃ 5.0Glucose and magnesium sulfate are sterilized separately. CaCO₃ isdry-heat sterilized at 180° C. for 2 h. The pH is adjusted to 7.0.

Example 16 Production of L-Ornithine by E. coli 382ΔargFΔargIΔyjjK

To test the effect of inactivation of the yjjK gene on L-ornithineproduction, the DNA fragments from the chromosome of the above-describedE. coli MG1655 ΔattBphi80 native ΔyjjK::Km^(R) are transferred to theL-ornithine producing E. coli strain 382ΔargFΔargI by P1-transduction toobtain the strain 382ΔargFΔargIΔyjjK. The strain 382ΔargFΔargI isobtained by consecutive deletion of argF and argI genes on thechromosome of 382 strain (VKPM B-7926) by the method initially developedby Datsenko K. A. and Wanner B. L. called “λRed/ET-mediated integration”(Datsenko K. A. and Wanner B. L., Proc. Natl. Acad. Sci. USA, 2000,97(12):6640-6645). According to this procedure, two pairs of PCR primershomologous to both the region adjacent to the argF or argI gene and thegene which confers antibiotic resistance in the template plasmid areconstructed. The plasmid pMW118-λattL-cat-λattR (WO 05/010175) is usedas the template in the PCR reaction.

Both E. coli strains, 382ΔargFΔargI and 382ΔargFΔargIΔyjjK, areseparately cultivated with shaking at 37° C. for 18 h in 3 mL ofnutrient broth, and 0.3 mL of the obtained cultures are inoculated into2 mL of a fermentation medium in 20×200-mm test tubes and cultivated at32° C. for 48 h on a rotary shaker.

After the cultivation, the amount of ornithine which accumulates in themedium is determined by paper chromatography using a mobile phaseconsisting of n-butanol:acetic acid:water=4:1:1 (v/v). A solution ofninhydrin (2%) in acetone is used as a visualizing reagent. A spotcontaining ornithine is cut out, ornithine is eluted with 0.5% watersolution of CdCl₂, and the amount of ornithine is estimatedspectrophotometrically at 540 nm.

The composition of the fermentation medium (g/L) is as follows:

Glucose 48.0 (NH₄)₂SO₄ 35.0 KH₂PO₄ 2.0 MgSO₄•7H₂O 1.0 Thiamine-HCl0.0002 Yeast extract 1.0 L-isoleucine 0.1 L-arginine 0.1 CaCO₃ 5.0Glucose and magnesium sulfate are sterilized separately. CaCO₃ isdry-heat sterilized at 180° C. for 2 h. The pH is adjusted to 7.0.

While the invention has been described in detail with reference topreferred embodiments thereof, it will be apparent to one skilled in theart that various changes can be made, and equivalents employed, withoutdeparting from the scope of the invention. All the cited referencesherein are incorporated by reference as a part of this application.

What is claimed is:
 1. A method for producing an L-amino acidcomprising: (i) cultivating the bacterium of the familyEnterobacteriaceae in a culture medium to produce said L-amino acid inthe bacterium and/or the culture medium, and (ii) collecting saidL-amino acid from the bacterium and/or the culture medium, wherein saidbacterium has been modified to attenuate expression of the yjjK gene. 2.The method according to claim 1, wherein said bacterium belongs to thegenus Escherichia.
 3. The method according to claim 2, wherein saidbacterium belongs to the species Escherichia coli.
 4. The methodaccording to claim 1, wherein said bacterium belongs to the genusPantoea.
 5. The method according to claim 4, wherein said bacteriumbelongs to the species Pantoea ananatis.
 6. The method according toclaim 1, wherein said expression is attenuated due to inactivation ofthe yjjK gene.
 7. The method according to claim 6, wherein said gene isdeleted.
 8. The method according to claim 1, wherein said the yjjK geneis encoded by the nucleotide sequence of SEQ ID NO: 1 or a variantnucleotide sequence of SEQ ID NO:
 1. 9. The method according to claim 1,wherein said L-amino acid is selected from the group consisting of anaromatic L-amino acid and a non-aromatic L-amino acid.
 10. The methodaccording to claim 9, wherein said aromatic L-amino acid is selectedfrom the group consisting of L-phenylalanine, L-tryptophan, andL-tyrosine.
 11. The method according to claim 9, wherein saidnon-aromatic L-amino acid is selected from the group consisting ofL-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline,L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine,L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-proline,L-serine, L-threonine, and L-valine.